Mucins in pancreatic cancer: A well‐established but promising family for diagnosis, prognosis and therapy

Abstract Mucins are a family of multifunctional glycoproteins that mostly line the surface of epithelial cells in the gastrointestinal tract and exert pivotal roles in gut lubrication and protection. Pancreatic cancer is a lethal disease with poor early diagnosis, limited therapeutic effects, and high numbers of cancer‐related deaths. In this review, we introduce the expression profiles of mucins in the normal pancreas, pancreatic precursor neoplasia and pancreatic cancer. Mucins in the pancreas contribute to biological processes such as the protection, lubrication and moisturization of epithelial tissues. They also participate in the carcinogenesis of pancreatic cancer and are used as diagnostic biomarkers and therapeutic targets. Herein, we discuss the important roles of mucins that lead to the lethality of pancreatic adenocarcinoma, particularly MUC1, MUC4, MUC5AC and MUC16 in disease progression, and present a comprehensive analysis of the clinical application of mucins and their promising roles in cancer treatment to gain a better understanding of the role of mucins in pancreatic cancer.


| INTRODUC TI ON
Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the United States and has a 5-year survival rate of <6%. 1 Pancreatic ductal adenocarcinoma (PDAC) remains the most common type, and nearly 250 000 new cases are diagnosed per year worldwide. 2 In China, it ranks sixth among all cancer types. 3 It is reported that the mortality rate of PC will become the second highest among cancer-related deaths before 2030. 4 PDAC is not yet fully understood by clinicians and scientists, and its major symptoms, including abdominal pain, obstructive jaundice and Cullen syndrome, are not easily detected at the early stages. Surgery is the only effective treatment for PDAC patients, but most patients miss the optimal time of surgery because of the late diagnosis. 5 Chemotherapy and radiotherapy may improve the poor prognosis slightly but with severe side effects. 3 Despite the increasing research efforts by cancer scientists, there is an urgent need for breakthroughs on the mechanism of oncogenesis and targeted therapy.
Patients diagnosed with locally advanced or metastatic disease usually have much higher levels of CA19-9. 2 Antigens such as CA19-9 are composed of oligosaccharide structures present on heavily glycosylated mucins born by the antigen. 6 Mucins are produced by various epithelial cells that are located on serine or threonine residues of the mucin core protein backbone. There are repetitive short stretches in the protein backbone termed tandem repeat regions (TRRs), while many O-glycosidic or N-glycosidic linkages are connected to the backbone. 7  Genes encoding mucins are denoted by the first three letters, "MUC," followed by the number in which they were discovered chronologically. 10 Mucins are divided into two categories: membrane-bound mucins and secreted mucins. The first class includes MUC1, MUC3, MUC4 and MUC16, which have been frequently investigated. MUC12, MUC13, MUC17 and MUC21 are also included in this group but limited studies have been performed on these. The protein structure of these mucins is composed of an N-terminal portion and a transmembrane domain, which has many phosphorylation sites that are involved in signal transduction. 10,11 The latter group of secreted mucins include MUC2, MUC5AC, MUC5B, MUC6 and MUC7, which are important for the formation of mucin heterodimers or homodimers. 9 Furthermore, another mucin, MUC18 (melanoma cell adhesion molecule), belongs to the immunoglobulin superfamily. 10 As well as protecting the barriers of mucous membranes, mucins also have important roles in cellular regeneration, differentiation, migration, adhesion and signalling. 8,12 Mucins may represent a diagnostic parameter for the early detection of PC and act as specific discriminated biomarkers among PC, pancreatic intraepithelial neoplasia (PanIN) and pancreatitis.
Our review aims to summarize and update the role of mucins in the pathogenesis of PC and focuses on the diagnostic, prognostic and therapeutic functions of these glycoproteins in PDAC.

| MUCIN S IN THE NORMAL PAN CRE A S
Exploring the distribution of certain mucins in normal pancreatic tissue may help to determine their biological significance. Much research has focused on mucin expression in normal and pathological pancreatic tissue to reveal a significant role in cancer pathogenesis. 11-14 MUC1 has been widely studied for many years because of its oncogenic features in various cancers including PC. 15 In normal pancreatic tissue, MUC1 is expressed in the intralobular ducts and centrally in the interlobular ducts, while the glycoforms of MUC1 are undetectable in the main pancreatic duct. MUC2 and MUC4 expression is also undetectable in the normal pancreas. 16 MUC3 expressed in the main pancreatic duct is reduced to low levels at 13 weeks of gestation, and MUC6 can be detected from 13 weeks in the pancreatic duct system, and is mainly detected in small interlobular ducts and developing acini, 7 and also in normal adult pancreas tissue. [16][17][18] Furthermore, while MUC5AC is not detectable, MUC5B is found in normal pancreatic tissue and the normal pancreatic duct, 5,13,14 unlike MUC7, which is also undetectable in the normal pancreas. 7 Other mucin genes, such as MUC11, MUC12 and MUC17 can be detected in normal pancreatic tissues. However, their specific expression pattern in the development of PC is unclear. 19,20 It was reported that there is relatively low expression of MUC20 and MUC21 in normal pancreatic tissue. 21,22

| MUCIN S IN PRECUR SOR LE S I ON S OF THE PAN CRE A S
Pancreatic ductal adenocarcinoma is the major type of pancreatic  11,24,25 Other studies revealed that MUC1 RNA levels are low in the tissue of both chronic pancreatitis and normal pancreas, while high MUC1 expression indicates the development of PC or PanIN progression. 5 As for mucin expression in intraductal papillary-mucinous neoplasm (IPMN), MUC1 tends to promote the development of PDAC and the potential for metastasis. 26 Similarly, MUC4 is minimally expressed in PanIN and chronic pancreatitis but is highly expressed in PC cell lines, 14,27 and aberrant overexpression of MUC4 was also associated with the progression of PanIN to PC, 28,29 while the intensity of expression increased in parallel with the severity of cellular dysplasia. 29 MUC5B is weakly expressed in pancreatic precursor lesions, while MUC5AC is gradually expressed during PanIN transformation. Since MUC5AC is seldomly expressed in the normal pancreas, 30 it has value as a biomarker of early neoplastic lesions, which correlate with poor prognosis. 31 Although MUC6 can be detected in the foetal pancreas in the early period of gestation, 32,33 it was reported have no involvement in the prognosis of PC patients. 34

| MUCIN S IN PAN CRE ATIC C AN CER
Mucins form a protective coat around cancer cells and have critical roles in the carcinogenesis of PC. 35 Several studies of the abnormal expression of mucins in PDAC indicated their potential roles in its pathogenesis, such as restricting intracellular drug uptake, facilitating immune escape, up-regulating signal pathways or even serving as prognostic biomarkers. [36][37][38] PDAC is characterized by the differential expression or glycosylation of the mucin family as it transforms from healthy tissue to cancer, as outlined in Table 1.

| MUC1
Many studies have focused on MUC1 as an oncogene, not only in PDAC but also in different cancer types. [39][40][41] MUC1 is expressed mainly along on the luminal cell surface and partially along the basolateral surface, and is detected in PDAC exhibiting aggressive behaviour. 42 High MUC1 expression is detected in more than 60% of PC cases and is significantly associated with tumour size. Furthermore, the higher the level of MUC1 expression, the poorer the prognosis in patients with PC. 43 In addition, PC manifests high expression rates of MUC1 glycoforms (MUC1/MY.1E12, 98%; MUC1/DF3, 96%; MUC1/HMFG1, 76%; and MUC1/CORE, 66%). 44 The cytoplasmic tail of MUC1 (MUC1-C) is widely involved in carcinogenesis and is correlated with PC progression by promoting the aggressive and metastatic phenotype. 45 MUC1-C contains multiple phosphorylated residues and interacts with signal transducers, cytokines, growth factors and other receptor tyrosine kinases to activate downstream signalling cascades. 46 For example, integrin-linked kinase, platelet-derived growth factor receptor-β and Met receptor tyrosine kinase modulate MUC1-C and enhance its oncogenic properties in PC. [47][48][49] Moreover, MUC1 in PC binds to β-catenin and EGFR to activate cell proliferation via the Wnt/ß-catenin or MAPK pathways. 50

| MUC4
MUC4 is overexpressed in most PCs, or even as early as the PanIN-I stages. 29,59 Several studies reported that MUC4 imparts several oncogenic properties and is detected in 32%-89% of PDAC patients. 28 Furthermore, MUC5AC seems to be a sensitive biomarker of early pancreatic neoplasms, providing another link with unfavourable prognosis. 30,31 In addition, the combination of MUC5AC and CA19-9 presented optimal performance and improved specificity compared with CA19-9 to differentiate PC from healthy controls. 73 expression was found to be remarkably lower in low-grade dysplasia compared with high-grade dysplasia, 78 while it was much higher in metastatic foci than that at primary sites; thus, we can conclude that it may have a pivotal function in the metastasis of PC. 79 Clinically, it is also combined with CA19-9 to evaluate the severity and prognosis of PC. 80,81 MUC16 is involved in many oncogenic signalling pathways, such as activating LMO2 and JAK2 to promote proliferation, and up-regulation of MUC16 stimulates mTOR and c-MYC to reprogram PC metabolism, enhancing glycolysis and cell proliferation. 82,83 MUC16 can also activate the AKT and MAPK pathways to promote metastasis, 83 and the MUC16-C terminal can promote the enrichment of Tregs through IL-6 activation of the JAK-STAT pathway in PC. 84 It was also reported that MUC16 might correspond with the up-regulation of other mucins, such as MUC1 and MUC4 in PC. 85 Overall, the involvement of MUC16 in the carcinogenesis of PC has been widely identified.

| Others
Mucins in pancreatic disease, particularly MUC1, -4, -5 and -16 have gained extensive attention because of their important roles in carcinogenesis, differential diagnosis and prognosis prediction.
Other mucin family members have also been studied in the field of PC. MUC3 has cysteine-rich domains that can inhibit apoptosis and enhance the progression of PC cells, 86 whereas MUC6 shows no effects on patient survival in PC. 34

| ROLE OF MUCIN IN THE C ARCINOG ENE S IS OF PAN CRE ATIC C AN CER
Although the oncogenic mechanism underlying pancreatic malignancy remains unclear, several studies have identified the critical role of mucins in PC formation, such as prompting tumorigenicity, enhancing metastasis and producing chemoresistance by O (or N)linked oligosaccharides. 37 In summary, mucin-mediated interactions, oncogenic signalling pathways and genetic alterations contribute to PC carcinogenesis.  95 and act as mediators of cellular adhesion during metastasis. 96 The pancreatic tumour microenvironment (TME) contains fibroblasts, pancreatic stellate cells, collagen, fibronectin and some cytokines, and importantly, mucin has many interactions with the TME to mediate immune evasion, oncogenic signalling and angiogenesis. 93,97   To further enhance the diagnostic value of CA19-9, it is combined with other mucins such as MUC5AC. 64,72 Additionally, endoscopic ultrasonography-guided, fine-needle aspiration (EUS-FNA) is also applied to the diagnosis before surgery as a minimally invasive method. MUC16 and MUC4 have high specificity in diagnosing PDAC from healthy controls with 63% and 67% sensitivity by EUS-FNA, respectively. 101 The latest research showed that MUC4 levels in the cystic fluid of IPMN patients could accurately discriminate high-from low-risk cystic neoplasms with high sensitivity. 102 107 Analysis of mucin expression as well as other biomarkers contributes to providing superior diagnostic information and enhanced monitoring of risk of disease recurrence.

| MUCIN S A S PROG NOS TI C IND I C ATOR S IN PAN CRE ATIC C AN CER
The overall survival curves associated with members of the mucin family were depicted by online Kaplan-Meier plotter (www.kmplot. com), a novel interactive website that aims to compare gene expression levels and prognostic conditions according to TCGA. The median was selected as the group cut-off value for survival plots.
Correlations between the mRNA levels of the mucin family and the overall survival of patients with PC is shown in Figure 2

| MUCIN -BA S ED THER APEUTI C S TR ATEG IE S
Mucins are involved in many malignant processes including evasion, invasion and metastatic by affecting oncogenic signalling, cell survival, proliferation and resistance to chemotherapeutics. 38,62 Furthermore, several mucins have been linked with tumour progression, chemoresistance and prognosis in PC. Because of these attributes, mucin-based therapy has also been applied for PC strategies including vaccines, antibodies, gene therapy and mucolytic agents ( Figure 3). which specifically targets MUC1, has been studied in PC. 118 Tumour targeting by 131 iodine (131I)-PAM4, 99mTechnetium (99mTc)-PAM4, and the humanized form of 90Y-PAM4 (h90Y-PAM4) was shown to be partially effective with acceptable adverse effects. 119,120 And an α-immunoconjugate of a monoclonal antibody, also known as C595, which can discern the TRR on MUC1, has been adopted as a valuable approach. 121 However, although some evidence for their effectiveness is promising, the side effects of radiotherapy such as neutropenia and thrombocytopenia as well as the high cost should be considered. 122

| Gene therapy
Several ongoing clinical studies proved that down-regulation of the expression of selected mucins could be a new therapeutic approach for PC. Knockdown of MUC1 by short interfering RNA inhibited the proliferation of PC cell lines, and injection of these cells into the pancreatic tissue of an animal model reduced the incidence of metastasis. 123 Similarly, MUC4 interference by siRNA in CD18/HPAF PC cells also inhibited proliferation in an orthotopic mouse model. 90 Except for targeting down-regulated mucins, the mucin gene promoter also will induce tumour cell death in MUC1expressing PC. 124 Although some studies showed the potential effect of gene therapies, they neglected to address the challenge of delivering the gene into the human body and are also not clinically approved.

F I G U R E 3
Mucins play important roles in the therapy of pancreatic cancer. The red arrow indicates the application of mucin-based vaccines. The blue arrow shows the antibody targeting mucins for therapy. The yellow arrow implicates the gene therapy by altering the expression of mucins. The green arrow manifests the mechanism of mucolytic agents

| Mucolytic agents
It has been reported that changes in the dense mucin mesh could alter the absorption of cytotoxic drugs. Mucins constitute a barrier on the cell surface that can infect drug uptake. 125 Therefore, drug-dissolving mucins such as bromelain and N-acetylcysteine (NAC) have been applied. 126,127 Bromelain is a cysteine proteinase composed of thiol endopeptidases that destroy the glycosidic bonds of mucins, while NAC denatures the disulphide bonds of mucus to decrease mucus viscosity, facilitating its clearance. 128 The combination of such two drugs can be used as an adjunct to cytotoxic drugs because of its effect on the lysis of mucin on the cell surface. 129

| CON CLUS IONS
Pancreatic adenocarcinoma is a worldwide challenge with high rates of incidence and mortality. Although many efforts have been made to identify the main mechanism of PC occurrence and development,

ACK N OWLED G EM ENTS
We thank H. Nikki March, PhD, from Liwen Bianji, Edanz Editing China (www.liwen bianji.cn/ac), for editing the English text of a draft of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.